P428: THE FATTY ACID ELONGASE ELOVL6 IS CRUCIAL FOR HEMATOPOIETIC STEM CELL ENGRAFTMENT AND LEUKEMIA PROPAGATION
Authors: Yusuke Kiyoki, Takayasu Kato, Sakura Kito, Takashi Matsuzaka, Shin Morioka, Junko Sasaki, Kenichi Makishima, Tatsuhiro Sakamoto, Hidekazu Nishikii, Naoshi Obara, Mamiko Sakata-Yanagimoto, Takehiko Sasaki, Hitoshi Shimano, Shigeru Chiba
Journal: HemaSphere (2023)

Background: Lipid metabolism plays an essential role in regulating stem and cancer cell function. The enzyme ELOVL6 converts C16 saturated and monounsaturated fatty acids (FAs) to C18 species, the most important step for the de novo synthesis of endogenous long chain FAs. Variations in carbon chain length or degree of desaturation of FA greatly contribute to lipid diversity. However, whether the lipid diversity significantly regulates hematopoietic cell biology remains unknown. Aims: To clarify how the changes of FA composition by Elovl6 gene disruption (E6KO) affect normal hematopoietic cell biology and leukemogenesis. Methods: We analyzed FA composition in bone marrow (BM) cells by gas chromatography. To assess the effects of Elovl6 loss on the mouse hematopoietic stem cell (HSC), we performed competitive repopulation assays (CRAs) using WT or E6KO BM cells. Next, to investigate the roles of Elovl6 in leukemogenesis, we infected lineage-negative and c-Kit+ cells from BM of WT or E6KO mice with MLL::AF9 (MA9) retrovirus and transplanted lethally irradiated mice with either WT or E6KO MA9 cells. Next, we performed RNA-seq and chemotaxis assay using WT or E6KO MA9 cells. Finally, we generated E6KO MA9 cells expressing Flag-tagged ELOVL6 (E6KO MA9Elo6) and mock controls (E6KO MA9mock), and performed transplantation. Results: To determine whether hematopoietic cells of E6KO mice exhibit changes in FAs composition, we analyzed FAs in bone marrow (BM) cell lysates. We found that the ratio of C18:0 to C16:0 as a marker for Elovl6 enzymatic activity was significantly decreased in E6KO cells compared to WT cells. In CRAs using WT or E6KO donor BM cells mixed at a 1:1 ratio with competitor BM cells, the chimerism of peripheral blood was about 50% in mice transplanted with WT cells but remained at < 0.2% in those with E6KO cells at 12 weeks after transplantation. In AML mouse model using MA9 cells, all mice receiving WT MA9 cells developed AML; however, AML propagation in E6KO MA9 cell-transplanted recipients was impaired. The results of RNA-seq and gene enrichment analysis showed that chemotaxis, CXCR4 pathways, PI3K pathways, and Rac1 pathways were enriched in E6KO MA9 cells, relative to WT MA9 cells. We tested the chemotaxis of E6KO CD34-, lineage-negative, Sca-1+ and c-Kit+ (CD34-LSK) and MA9 cells toward CXCL12 in a migration assay. A greater fraction of WT CD34-LSK and MA9 cells than that of these E6KO cells migrated to the CXCL12-containing chamber. We then examined the pharmacologic effects of inhibiting ELOVL6 and the PI3K pathway on chemotaxis. In a migration assay, Compound B, an ELOVL6 inhibitor, pan-PI3K inhibitor Copanlisib, or pan-RAC inhibitor EHT-1864 decreased the migration rate in WT MA9 cells, but not significantly in E6KO MA9 cells, migration of which was already compromised. By contrast, the AKT inhibitor, MK2206, did not alter WT or E6KO MA9 cell migration. We thus investigated whether Rac1 activation was blocked by Elovl6 loss. Levels of GTP-bound Rac1 markedly increased in WT MA9 cells following CXCL12 stimulation, but the increase was not significant in E6KO MA9 cells. Thus, ELOVL6 may regulate a CXCL12-CXCR4 pathway, resulting in PI3K-Rac1-mediated chemotaxis. Finally, to determine whether exogenously expressed Elovl6 could rescue E6KO MA9 cell phenotypes, we transplanted lethally irradiated syngeneic mice with either E6KO MA9mock or E6KO MA9Elo6 cells. Among E6KO MA9mock transplanted mice, few recipients developed AML, while 10 out of 14 recipients had died due to AML after E6KO MA9Elo6 cell transplant. Summary/Conclusion: ELOVL6 is essential for HSC engraftment after BM transplantation, and Elovl6 loss blocks AML development in a moue model. Altering lipid diversity could be a potential strategy to treat AML. Keywords: Lipid metabolism, Acute myeloid leukemia, Leukemogenesis